While the observation of electrophoretic phenomena was made at least as early as 1807 by the Russian physicist F. F. Reuss, it was not until the late 1930's that scientific applications of this technique came to see employment. This was largely on account of the work of Arne Tiselius.
Recognizing that in the migration of a particle through an electrophoretic material under the influence of an electric field, velocity is a function not only of field strength, but also certain of the particle's other characteristics, Tiselius built a cell using what is now referred to as the moving boundary method and provided it with a solution of plasma, postulating that plasma was not a homogeneous material. The experiment revealed that human plasma does indeed consist of a heterogeneous solution of materials, and identified for the first time alpha, beta and gamma globulins, each of which have different electrophoretic mobilities. As a consequence of this work, electrophoresis has been extensively used for many years whenever it is desired to separate solutions of proteins and other macromolecules.
Notwithstanding such widespread and longstanding use, the technique and apparatus of electrophoresis remains today much the same as it was in the time of Tiselius. Perhaps the only major change in the technology has been the introduction of gel as an electrophoretic medium.
Today, gel matrix electrophoresis is by far the most common form of electrophoresis. Gel matrices exhibit superior characteristics on account of their ability to substantially reduce to zero convective phenomena, which otherwise would distort the path of particles in the medium. Such convective phenomena introduce extremely large errors into the process in the case of conventional media. In addition, the use of gel supporting media also greatly reduces mixing of the various parts of the sample, and therefore provides for more stable separation.
Generally, electrophoretic mobility is a function of molecular size, molecular charge and molecular shape. It has been suggested that the molecular structure of the gel also provides a molecular sieving effect, resulting in greater resolving power where the molecules undergoing electrophoretic separation have different sizes. In addition, gels allow for the practical employment of gradients of pH and of reagents. Typically, such gradients are used in two dimensional arrays to provide a greater range of resolution.
For example, a widely employed use of a gel supporting media takes advantage of the incorporation into the gel matrix of a large amount of detergent which has the effect of denaturing or unbundling relatively tightly clustered protein molecule structures, to transform them into long flexible polymeric chains to which detergent molecules are bound. In this case, if the chains of the polymeric chains are of various lengths, even if they have uniform molecular characteristics, they will be caused to move through the electric field in the gel medium with a velocity which is determined by the length of the particular polymer. Since molecular weight is a function of polymer length, this method is used in a wide variety of applications where molecular weight is to be determined.
Typically, electrophoresis is carried out today in modular cells which comprise a layer of gel material, sandwiched between a pair of flat plastic or glass plates. Such cells contemplate the simultaneous and parallel running of a plurality of electrophoretic separation operations, each of which is carried out in its own elongated portion of the gel.
Typically a sample consisting of a weighted buffer solution and a known mixture of materials is used as a standard for the plurality of electrophoretic separations to be carried out simultaneously and in parallel with the separation of the known marker, all within the same cell and under the influence of the same electric field. Thus, a plurality of wells are cast into one end of the layer of gel and a known sample or marker, for example, of buffer and proteins is inserted into the first well. Unknown solutions of materials in buffer are then inserted into the other wells and an electric field applied across the cell. The result is migration of materials along parallel paths extending from the wells which are disposed side by side in the gel layer. After electrophoretic separation has been preformed, a dye is used to stain the separated materials, resulting in the revelation, typically, of a series of dark bands along each of the paths of electrophoretic separation.
By comparison of an unknown material to one or more electrophoretic separations contained within, typically, the same cell and of known characteristics, the characteristics of the unknown band may be determined by comparison to the marker. One typical application of such an operation is in the forensic field. For example, if a sample protein taken at the scene of a crime is known to consist of materials which may be electrophoretically separated, samples of similar proteins may be taken from suspects and the existence or nonexistence of a match used to determine the unique identity of the person from which the unknown sample originated because of the uniqueness of proteins in biosystems.
In accordance with the preferred embodiment, in order to provide parallel separation of input material for electrophoresis, it is necessary that the gel be formed with a plurality of input wells. In accordance with current practice, this is achieved by forming the gel with the desired wells. However, a problem is presented because of the fragile nature of the gel material. In particular, the wells cast into the material are likely to break and loose their integrity during handling, transport, packaging or the like. Thus, the common means employed by the industry to maintain structural integrity is to cast the wells using a comb. However, this comb is a relatively expensive item and thus raises the per unit cost of the electrophoretic cell. In addition, the use of the comb also complicates assembly requiring additional effort and expense during the same.
The use of the comb, which is generally cut from a planar sheet of material, presents an interesting problem for optimization in the design of the electrophoretic cell. In particular, a comb must have the same thickness as the gel medium in which electrophoresis is being carried out. If this were not the case, the possibility would exist for the migration of sample into the sides of the well if, for example, the comb were thinner than the gel. This would result in the substantial waste of extremely large amounts of what may be a very precious sample. Indeed, such a possibility cannot be tolerated. Rather, the interface between the sample in the solution and the electrophoretic gel medium must be strictly the intersection of a plane perpendicular to the gel medium and the gel medium. Ideally, the thinnest gel medium is desirable, at least from the standpoint of separation of the sample consistently and also from the standpoint of ease of transfer of the sample to a permanent record, typically made of cellulose material. Such transfer is achieved by placing the finished electrophoretic separation against the medium to receive the transfer and applying an electrical field perpendicular to that applied during electrophoresis. This results in driving the sample matrix out of the gel and into the cellulose or other transfer media for permanent storage. However, because of the nature of media and gels and the electrical fields which can be created therein, such transfers are not easily or well achieved in the case of thicker gels. In addition, thinner gels have the advantage of allowing faster electrophoretic separation. Thus, the design of a most effective and easy to use electrophoretic system specifies extremely thin gels. The thinness of these gels, of course, varies for the various gel media involved and the application desired.
While the above would appear to be a straightforward approach to the design of an ideal cell, unfortunately, because of the fact that the interface between the sample solution and the gel edge must be a clean, small area equal in thickness to the thickness of the gel, a situation is created in which a thinner gel creates problems which would suggest that the gel should be as thick as possible. More particularly, during electrophoresis, it is desirable to have as large a surface area as possible for entry of the sample into the electrophoretic gel. Naturally, in order for such an area to be made, the thickness of the gel must be increased. The large area is required in order to provide for the entry of the sample into the edge of the medium in a relatively short period of time. However, increasing the thickness of the medium creates the other problems discussed above. At present, electrophoretic cell design involves an optimization of both factors, recognizing that improving the operation of the system from one standpoint results in decreasing effectiveness of other parameters of the electrophoretic system.
In an attempt to overcome this problem some cells have been manufactured in which the combs are relatively tall, resulting in the production of tall, thin reservoirs for holding sample in buffer solutions. However, the use of a tall reservoir results in large distances for a certain portion of the sample to transverse on its way from, for example, the top of the reservoir to the interface between the sample-buffer solution and the electrophoretic gel. This results in increasing the time over which a particular constituent of the sample buffer solution will be transferred into the gel media and thus the thickness of the band which is made by the system at the end of the electrophoretic separation process. Thus, the use of tall combs and tall reservoirs does not provide an acceptable solution.